Madhadron:SpheroplastingSmegmatis: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 30: | Line 30: | ||
===Notes=== | ===Notes=== | ||
Need to measure mycolic acid concentration to make sure that they really have sloughed off cell wall. | Need to measure mycolic acid concentration to make sure that they really have sloughed off cell wall (Mike Glickman is the local expert). | ||
Revision as of 11:51, 15 December 2006
(Based on Udou et al, 1982)
- Grow culture of smeg overnight
- Add 20% glycine to final concentration 1.2%, and incubate for 16-20h.
- Centrifuge at 1,200g for 15 minutes, decant supernatant. Resuspend in same volume SMM.
- Centrifuge again. Resuspend in same volume of P medium.
- Incubate for 16 to 20h
- Centrifuge at 300g for 7 minutes and discard pellet.
- Centrifuge supernatant at 1,200g for 15 minutes.
- Wash twice with SMM
P medium
(Additives per L of water)
- 120g sucrose
- 10g glucose
- 12g L-glycine
- 1g (NH_4)_2 SO_4
- 0.25g K_2 SO_4
- 2.03g MgCl_2 times 6H_2)
- 3.68 CaCl_2 * 2H_2O
- 0.1g KH_2 PO_4
- 0.025M [N-tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES; pH 7.2)
- 50mg lysozyme
- 30mg lytic enzyme no.2
SMM
0.5M sucrose + 20mM MgCl_2 + 0.02M maleate (pH 6.5)
Notes
Need to measure mycolic acid concentration to make sure that they really have sloughed off cell wall (Mike Glickman is the local expert).
